Spectroscopic Characterization of Collagen Cross-Links in Bone

TLDR

Collagen, the most abundant protein in mineralizing tissues, relies on its cross‑linking pattern to confer tensile strength and viscoelasticity to the fibrillar matrix. The authors applied Fourier‑transform infrared spectroscopy and imaging to diverse collagen samples, resolving the amide I region into subbands at ~1660 and ~1690 cm⁻¹ whose area ratios reflect pyridinoline and dehydrodihydroxylysinonorleucine cross‑links. The study demonstrates that this spectroscopic parameter can map Pyr and DHLNL distribution in mineralized tissues, enabling calculation of relative cross‑link amounts in ~5 µm calcified sections with ~7 µm spatial resolution.

Abstract

Collagen is the most abundant protein of the organic matrix in mineralizing tissues. One of its most critical properties is its cross-linking pattern. The intermolecular cross-linking provides the fibrillar matrices with mechanical properties such as tensile strength and viscoelasticity. In this study, Fourier transform infrared (FTIR) spectroscopy and FTIR imaging (FTIRI) analyses were performed in a series of biochemically characterized samples including purified collagen cross-linked peptides, demineralized bovine bone collagen from animals of different ages, collagen from vitamin B6-deficient chick homogenized bone and their age- and sex-matched controls, and histologically stained thin sections from normal human iliac crest biopsy specimens. One region of the FTIR spectrum of particular interest (the amide I spectral region) was resolved into its underlying components. Of these components, the relative percent area ratio of two subbands at approximately 1660 cm(-1) and approximately 1690 cm(-1) was related to collagen cross-links that are abundant in mineralized tissues (i.e., pyridinoline [Pyr] and dehydrodihydroxylysinonorleucine [deH-DHLNL]). This study shows that it is feasible to monitor Pyr and DHLNL collagen cross-links spatial distribution in mineralized tissues. The spectroscopic parameter established in this study may be used in FTIRI analyses, thus enabling the calculation of relative Pyr/DHLNL amounts in thin (approximately 5 microm) calcified tissue sections with a spatial resolution of approximately 7 microm.